The invention relates to compositions and methods for altering fertility and more specifically to sperm-specific forms of the catalytic subunit (referred to as xe2x80x9cCxe2x80x9d) of cAMP-dependent protein kinases.
The cAMP-dependent protein kinase (PKA) is a major enzyme in cellular signal transduction and is thought to mediate most of the physiological responses to cAMP in eukaryotic cells. Below a cAMP threshold concentration, PKA exists as an inactive tetramer of two catalytic (C) subunits and two regulatory (R) subunits that together can be represented as CR2C. The two R subunits form a dimer with each protomer attaching to the substrate-binding site of a C subunit. Some isoforms of R also associate with binding proteins collectively termed A-kinase anchoring proteins; it is believed that through these interactions PKA is targeted to specific subcellular compartments.
Activation of adenylate cyclase by extracellular signals raises the intracellular concentration of cAMP, and at a certain threshold concentration cAMP binds to the R subunits of the PKA tetramer, releasing C to phosphorylate its substrates.
There are three known genes encoding mammalian C. The Cxcex1 gene appears to be expressed in most tissues, including the brain, while Cxcex2 gene expression is detected mainly in the brain. Cxcex3 is a transcribed retroposon that has been found in primates and whose expression is detected only in testis. Proteins expressed from recombinant clones of Cxcex1, Cxcex2, and Cxcex3 have been shown to have distinct biochemical properties.
cAMP-dependent signaling has an important role in the control of sperm movement. Mammalian sperm are nonmotile in the testis, but as they pass through the epididymis they acquire the capacity for motility. This process is known as xe2x80x9cepididymal maturationxe2x80x9d and is essential for the sperm to fertilize an egg. Several studies have shown that changes in sperm cAMP levels are involved in epididymal maturation. The mechanisms by which alterations in cAMP levels lead to epididymal maturation are largely unknown.
The invention is based in part on the discovery of a novel sperm-specific form of the catalytic subunit of cAMP-dependent protein kinase referred to herein as Cs polypeptide, or just Cs, which is an alternatively spliced transcript of the Cxcex1 gene. Cs is unique in that it has an acetylated amino terminus with an N-terminal amino acid sequence of six amino acids, which differs from the N-terminal sequence of all other previously described forms of C. The remainder of the amino acid sequence of Cs is identical to that of Cxcex11, another protein encoded by the Cxcex1 gene, which was formerly referred to as the Cxcex1 protein.
The region of identity begins with the amino acids encoded by the beginning of the second exon of Cxcex1. Thus, Cs arises as a result of testis-specific splicing of an exon encoding the novel N-terminus to the remaining Cxcex1 exons. The sequence identity of the amino acids beginning with the second exon holds true in a wide variety of mammals. For example, the remainder of the sequence of ovine Cs is identical to that of ovine Cxcex11, and the remainder of the sequence of human Cs is identical to that of human Cxcex11.
Because Cs is expressed only in testis cells (i.e., male cells), it provides a unique target for the diagnosis and therapy of fertility disorders, as well as a basis for novel male contraceptives and fertility enhancers. Thus, for example, nucleic acids encoding Cs polypeptides, and agents based on these sequences, can be used for diagnosing and treating conditions associated with spermatocyte function, e.g., in the promotion or inhibition of fertility.
In general, the invention features isolated nucleic acids encoding Cs polypeptides. For example, an isolated nucleic acid may encode a polypeptide comprising a first peptide linked to a second peptide, wherein the first peptide has the sequence Xaa1, Ser Xaa2 Xaa3 Xaa4 Asp (SEQ ID NO:1), where Xaa can be any amino acid, and wherein the second peptide is at least 85%, 90%, 95%, 98%, 99% or even 100% identical to a peptide having the amino acid sequence of the predicted human Cxcex11 amino acid sequence beginning at exon 2 (SEQ ID NO:2). This sequence is shown in FIG. 1A as part of the full amino acid sequence of the human Cxcex11 polypeptide (SEQ ID NO:4). The amino acid sequence corresponding to SEQ ID NO:2 begins after the xe2x80x9c/xe2x80x9d. The nucleic acid can also encode the amino acid sequence of SEQ ID NO:2 wherein Xaa1 is Ala, Xaa2 is Asn or Ser, Xaa3 is Ser or Pro, and Xaa4 is Asn or Ser.
FIG. 1B shows the full nucleic acid sequence of human Cxcex1 (SEQ ID NO:5), including the portion beginning at exon 2 (after the xe2x80x9c/xe2x80x9d) and ending with nucleotide 1133 (SEQ ID NO:3). Both the amino acid and nucleotide sequences of human Cxcex1 are available from GenBank at Accession No. X07767.
In other embodiments, the second peptide is at least 75% or more, e.g., 100%, identical to the Cxcex11 amino acid sequence beginning at exon 2 of other animals, e.g., mammalian, species of interest, such as dog, cat, horse, cow, or pig. Thus, the Cs polypeptide can be designed to be species-specific, and can then be used to treat that particular species.
The amino acid sequence of the first peptide can include, e.g., Ala Ser Asn Pro Asn Asp (SEQ ID NO:6), which corresponds to the amino terminal amino acid sequence in mature ovine Cs, Pro Ser Ser Ser Asn Asp (SEQ ID NO:7), which corresponds to a predicted amino acid sequence encoded by a mouse pseudogene. The amino acid sequence of the first peptide can also include, e.g., Ala Ser Asn Ser Ser Asp (SEQ ID NO:46) or Ala Ser Ser Ser Asn Asp (SEQ ID NO:47). The latter sequences correspond to the amino terminal amino acid sequence in processed human and mouse Cs, respectively.
The nucleic acid encoding the first peptide can optionally encode the amino acid Met at the amino terminus, i.e., Met Xaa Ser Xaa Xaa Asn Asp (SEQ ID NO:24), Met Ala Ser Asn Pro Asn Asp (SEQ ID NO:8), Met Pro Ser Ser Ser Asn Asp (SEQ ID NO:9), Met Ala Ser Asn Ser Ser Asp (SEQ ID NO:36), or Met Ala Ser Ser Ser Asn Asp (SEQ ID NO:40).
The first peptide can thus be encoded by, e.g, 5xe2x80x2-GCTTCCAACCCCAACGAT-3xe2x80x2 (SEQ ID NO:10), 5xe2x80x2-CCTTCCAGCTCCAATGAT3xe2x80x2 (SEQ ID NO:11), 5xe2x80x2-ATGGCTTCCAACCCCAACGAT-3xe2x80x2 (SEQ ID NO:12), and 5xe2x80x2-ATGCCTTCCAGCTCCAATGAT-3xe2x80x2 (SEQ ID NO:13), 5xe2x80x2-GCTTCCAACTCCAGCGAT-3xe2x80x2 (SEQ ID NO:48), 5xe2x80x2-GCTTCCAGCTC-CAACGAT-3xe2x80x2 (SEQ ID NO:49), 5xe2x80x2-ATGGCTTCCAACTCCAGCGAT-3xe2x80x2 (SEQ ID NO:37), and 5xe2x80x2-ATGGCTTCCAGCTCCAACGAT-3xe2x80x2 (SEQ ID NO:41), which encode the amino acids of SEQ ID NOS: 6-9, 46-47, 36, and 40, respectively.
The isolated nucleic acid can also include a nucleic acid encoding ovine Cs (SEQ ID NO:14) as shown in FIGS. 2A-C. The predicted amino acid sequence of ovine Cs (SEQ ID NO:15) is also shown in FIGS. 2A-C.
In other embodiments, an isolated nucleic acid can encode a polypeptide comprising a first peptide linked to a second peptide, wherein the first peptide has the sequence Met Ala Ser Asn Ser Ser Asp (SEQ ID NO:36) or Ala Ser Asn Ser Ser Asp (SEQ ID NO:46), and the second peptide is at least 85%, 90%, 95%, 98%, 99% or even 100% identical to a peptide having the amino acid sequence of the predicted human Cxcex11 amino acid sequence beginning at exon 2 (SEQ ID NO:2). An example of such a nucleic acid is a nucleic acid encoding the human Cs polypeptide (SEQ ID NO:34), e.g., the nucleic acid of SEQ ID NO:35.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5xe2x80x2 end and one on the 3xe2x80x2 end) in the naturally occurring genome of the organism from which it is derived. Thus, a recombinant nucleic acid could include some or all of the 5xe2x80x2 non-coding (e.g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, such as a retrovirus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence. The term xe2x80x9cisolated Cs nucleic acidxe2x80x9d does not include Cs nucleic acid mixed or otherwise associated with other, closely related C nucleic acids, e.g., those that migrate at a similar rate as Cs on an electrophoresis gel.
By xe2x80x9clinkedxe2x80x9d is meant that a first portion, e.g., a peptide, is connected to a second portion, e.g., another peptide, by a covalent bond, e.g., a peptide bond, or by a non-covalent or other type of bond.
In some embodiments, the nucleic acid encoding a Cs polypeptide hybridizes under stringent conditions to a sequence complementary to 5xe2x80x2-ATGGCTTCCAACCCCAACGAT-3xe2x80x2 (SEQ ID NO:12), 5xe2x80x2-ATGCCTTCCAGCTCCAATGAT-3xe2x80x2 (SEQ ID NO:13), 5xe2x80x2-ATGGCTTCCAACTCCAGCGAT-3xe2x80x2 (SEQ ID NO:37), and 5xe2x80x2-ATGGCTTCCAGCTCCAACGA-3xe2x80x2 (SEQ ID NO:41).
Hybridization under xe2x80x9cstringent conditionsxe2x80x9d using short Cs-specific oligonucleotide probes, e.g., probes having the nucleotide sequence of, or the sequence complementary to, the nucleic acids encoding the first peptide in Cs, e.g., of SEQ ID NOs. 1, 4, 7-9, 36, 38, 40, 46, or 47 is hybridization at 37xc2x0 C. in 4xc3x97SSPE, 0.2% SDS, 0.5% non-fat dry milk and washing at 37xc2x0 C. in 2xc3x97SSC, 0.2% SDS after hybridization. Such Cs-specific oligonucleotide probes are used under stringent conditions to determine whether a sample or unknown nucleic acid is a Cs nucleic acid, i.e., a nucleic acid that encodes a Cs polypeptide. If the probe hybridizes to an unknown nucleic acid under stringent conditions, then the unknown nucleic acid is a Cs nucleic acid. Such Cs nucleic acids encode polypeptides having some or all of the biological activities possessed by naturally-occurring Cs. The biological activity can be measured using one of the assays described herein.
Nucleic acids with even closer matches to the probe sequence can be identified by performing post-hybridization washes in 2xc3x97SSC, 0.2% SDS at 42xc2x0 C., 45xc2x0 C., 50xc2x0 C., 55xc2x0 C., or even 65xc2x0 C.
The xe2x80x9cidentityxe2x80x9d of a nucleic acid or amino acid sequence can be measured using sequence analysis software (Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705) set to the default parameters therein. By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid having a sequence that is at least 85% identical to the sequence of the reference amino acid or nucleic acid sequence as measured using the sequence analysis software Package set to default parameters. Other polypeptides or nucleotide sequences can be more or less xe2x80x9cidentical,xe2x80x9d e.g., 90%, 95%, 98%, or even 99% or 100% identical.
For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
A suitable antisense oligo for mouse Cs is one that corresponds to nucleotides xe2x88x92187 to +16 of the nucleic acid encoding mouse Cs, and an antisense oligo for human Cs is one that corresponds to nucleotides xe2x88x92140 to +16 of the nucleic acid sequence encoding human Cs.
In another aspect, the invention features an isolated nucleic acid comprising a nucleotide sequence complementary to all or a portion of the nucleotide sequence encoding a Cs polypeptide, e.g., a nucleic acid complementary to the nucleic acid encoding the peptide of SEQ ID NO:1, SEQ ID NO:36, SEQ ID NO:40, or SEQ ID NO:46. Also included is an isolated nucleic acid complementary to a region of the 5xe2x80x2 untranslated region (UTR) of the Cs specific exon, e.g., nucleotides xe2x88x9229 to +22 (SEQ ID NO:20) in FIGS. 2A-C, which contains ovine 5xe2x80x2UTR sequences and exon 1 coding sequences, or nucleotides xe2x88x9229 to xe2x88x921 (SEQ ID NO:21) in FIGS. 2A-C, which contains only 5xe2x80x2UTR sequences. Alternatively, the isolated nucleic acid may be complementary to a region including both 5xe2x80x2 UTR and exon 1, e.g., nucleotides xe2x88x9220 to +22 (SEQ ID NO:22) or nucleotides xe2x88x925 to +15 (SEQ ID NO:23) of FIGS. 2A-C. Also included is an isolated nucleic acid complementary to the nucleic acid encoding the polypeptide of SEQ ID NO:1 with a Met on its amino terminal end, i.e., Met Xaa1 Ser Xaa2 Xaa3 Xaa4 Asn Asp (SEQ ID NO:2). Thus, the nucleic acid can include a nucleic acid of SEQ ID NOs:16-19, which are nucleic acids complementary to the nucleic acids of SEQ ID NOS: 10-13, respectively.
In another aspect, the invention features a nucleic acid having at least 12, preferably at least 15, 18, 25, or 50 contiguous nucleotides that are at least 85% identical to nucleotides in the sequence complementary to nucleotides xe2x88x9229 to +22 of ovine Cs (SEQ ID NO:20), murine Cs (SEQ ID NO:55), or human Cs (SEQ ID NO:56).
The invention also features an isolated enzymatic RNA molecule, e.g., a ribozyme, that specifically cleaves Cs RNA.
In another aspect, the invention includes a vector, e.g., an expression vector, that includes a nucleic acid molecule encoding a Cs polypeptide. The vector can be, for example, a plasmid or a virus. The vector may optionally include a regulatory element.
The invention also includes a host cell, e.g., a non-human mammalian cell, which contains the nucleic acid encoding a Cs polypeptide. Thus, the host cell can include a vector having a nucleic acid encoding Cs.
The invention also features substantially pure Cs polypeptides. By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length, and thus includes polypeptides, proteins, and peptides. A Cs polypeptide must include at least an acetylated N-terminal end, and the six amino acid peptide of SEQ ID NOs:1, 6, 8, 9, 24, 36, 40, 46, or 47 at its N-terminal end.
By xe2x80x9csubstantially purexe2x80x9d is meant a preparation which is at least 60% by weight (dry weight) the compound of interest, i.e., a Cs polypeptide, and which does not include any non-Cs polypeptides that have essentially the same molecular weight as Cs, e.g., Cxcex11 polypeptides. Thus, a substantially pure Cs polypeptide is not mixed or otherwise associated with proteins, such as other C proteins, that migrate at a similar position in an SDS-acrylamide gel. A preparation of a substantially pure Cs polypeptide is also substantially free of an antibody or any other compound that binds to a Cs polypeptide.
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions can be conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. The resulting substituted polypeptide should have some or all of the function of the non-substituted polypeptide. The substitutions are preferably in the second peptide in the full-length Cs polypeptide.
Preferably the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight of the Cs polypeptide. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, MALDI-TOF mass spectrometry (MS), or HPLC analysis.
The polypeptides of the invention include, but are not limited to, full-length Cs proteins or polypeptides, e.g., a polypeptide having the amino acid sequence of human Cs (SEQ ID NO:34), murine Cs, (SEQ ID NO:38), ovine Cs (SEQ ID NO:15), as well as modified forms of these polypeptides wherein the encoded amino terminal methionine is absent, and forms in which the amino terminal methionine is absent and the resulting amino terminal amino acid is acetylated.
Also included in the invention are peptides, e.g., a peptide having the amino acid sequence of SEQ ID NO:1, such as the peptides of SEQ ID NOS:6, 7, 8, 9, 24, 36, 40, 46, and 47. The invention also includes modified forms of these peptides in which the amino terminus is acetylated. Some of these peptides have, or can have, an amino terminal methionine residue.
The invention further includes recombinant polypeptides, natural polypeptides, and synthetic polypeptides, as well as polypeptides which are preproteins or proproteins.
Also included in the invention are modified forms of Cs polypeptides or Cs peptides, e.g., an acetylated or phosphorylated Cs polypeptide or peptide.
The Cs polypeptides of the invention can be expressed fused to another polypeptide, e.g., a marker polypeptide or fusion partner. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein or a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
By xe2x80x9cmature Csxe2x80x9d is meant a polypeptide having the sequence of full-length, wild type Cs of a specific animal species, e.g., the ovine Cs polypeptide of SEQ ID NO:15 or human Cs polypeptide of SEQ ID NO:34.
Also within the invention are soluble fusion proteins in which a full-length form of Cs or a portion (e.g., one or more domains) thereof is fused to an unrelated protein or polypeptide (i.e., a fusion partner) to create a fusion protein. The Cs polypeptide may be recombinantly produced, e.g., from expression of an isolated Cs nucleic acid.
In other embodiments, the invention features a substantially pure polypeptide which includes a first portion linked to a second portion, the first portion including a Cs polypeptide and the second portion including a detectable marker.
The invention further features an antibody (e.g., a monoclonal or polyclonal antibody) that specifically binds to a Cs polypeptide. The antibody can be raised against full-length Cs polypeptides, or Cs peptides such as one including an amino acid sequence corresponding to those at the amino terminus of the Cs polypeptide, e.g., peptides including the amino acid sequences of SEQ ID NO: 6, 7, 46, or 47. The antibody can also be raised to an acetylated peptide, e.g., an acetylated peptide including the amino acid sequences of SEQ ID NO: 6, 7, 8, 9, 24, 36, 40, 46, or 47. A Cs-specific antibody is useful as a specific marker for detecting testis (germ) cells and so can be used, e.g., to identify tumor tissue as derived from testis cells, e.g., metastasized from the testes.
The invention further includes a pharmaceutical composition comprising, e.g., a Cs polypeptide or peptide or Cs nucleic acid.
In another aspect, the invention includes a method for producing a Cs polypeptide by culturing a host cell containing a Cs nucleic acid molecule under conditions in which the nucleic acid molecule is expressed.
Also included in the invention is a method for detecting a Cs polypeptide in a sample by obtaining a biological sample, contacting the biological sample with an antibody that specifically binds to Cs under conditions that allow the formation of Cs-antibody complexes, and detecting the complexes (if any) as an indication of the presence or activity of Cs in the sample.
An antibody that xe2x80x9cspecifically bindsxe2x80x9d to an antigen is an antibody that recognizes and binds to a particular antigen, e.g., a Cs polypeptide, but that does not substantially recognize or bind to other molecules or proteins in a sample, e.g., a biological sample, which includes Cs.
The invention features a method of identifying a compound that modulates the expression or activity of Cs by comparing the expression or level of activity of Cs in a cell in the presence and absence of a selected compound, wherein a difference in the level of activity in the presence and absence of the selected compound indicates that the selected compound modulates the activity of Cs.
In addition, the invention features a method of treating a patient suffering from a disorder associated with aberrant expression or function of Cs (e.g., excessive expression or activity of Cs, or insufficient expression or activity of Cs) by administering to the patient a compound which modulates the activity or expression of Cs (e.g., inhibits expression or activity of Cs in a patient having excessive expression or activity of Cs, or increases the expression or activity in a patient having insufficient Cs expression or activity). Disorders associated with aberrant expression of Cs can include, e.g., fertility disorders as described herein.
The invention also includes a method of diagnosing a disorder associated with aberrant (e.g., decreased) expression or activity of Cs by obtaining a biological sample from a patient and measuring Cs expression in the biological sample. Increased or decreased Cs expression or activity in the biological sample compared to a control indicates that the patient suffers from a disorder associated with aberrant expression or activity of Cs.
Another aspect of the invention includes a method of inhibiting fertility by administering to a male in need thereof an effective amount of an antagonist of a Cs polypeptide or an antagonist of a Cs nucleic acid. For example, the invention includes a method of reducing levels of Cs, by administering to a male in need thereof an antibody to Cs, or an antibody to a Cs peptide. As another example, the invention includes a method of inhibiting fertility by administering to a male in need thereof an effective amount of an antisense oligonucleotide that inhibits expression of Cs. The antisense oligonucleotide is complementary to a nucleic acid sequence encoding Xaa Ser Xaa Xaa Asn Asp (SEQ ID NO:1), e.g., nucleic acid sequences of SEQ ID NOs:16-19, 41, 48, and 49. In other embodiments, the antisense oligonucleotide is complementary to the nucleic acid of SEQ ID NO:37 or SEQ ID NO:39.
The invention also includes a method of reducing levels of Cs by administering to a male in need thereof an enzymatic RNA molecule which specifically cleaves Cs RNA, e.g., in the portion of the nucleic acid sequence encoding SEQ ID NOs: 1, 6, 46, and 47, and others described herein.
In another aspect, the invention includes method of promoting fertility by administering an effective amount of a Cs polypeptide or a biologically active Cs peptide to a male in need thereof.
Also included is a method of promoting sperm motility by administering an effective amount of a Cs polypeptide or a biologically active fragment of a Cs polypeptide to a male in need thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict in terminology, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.